Carbonic anhydrase (CA) represents the second most abundant protein present inside erythrocytes. With the exception of the hagfish and other lampreys, carbonic anhydrase activity or content is normally extremely low in vertebrates in the plasma. Carbonic anhydrase exists in at least twelve different isozymes, with variable distribution among tissues. The CA-I and CA-II isozymes predominate in erythrocytes, with a small amount of CA-III. Isozymes CA-IV to XII are found in many other tissues, including the gut, kidney and lung. CA-IX is under study as a potential biomarker of renal and squamous cell carcinoma.
However, the characteristics and fate of free CA in plasma are poorly understood. Free CA-I, CA-II, and CA-III in plasma is rapidly bound and inactivated by a transferrin-like protein, followed by clearance in the reticuloendothelial system. Studies of radiolabelled CA in rats demonstrates an approximate half-life of two hours with elimination in the kidney and liver for CA isozymes I and II.
The activity of carbonic anhydrase can be measured by colorimetric assays, typically employing a substrate that is recognized and cleaved by the active site of CA. One such example is the compound para nitro phenylacetate. Since carbonic anhydrase has intrinsic esterase activity, it will cleave the dye (p-nitrophenol) from the acetate liberating a color in aqueous solution that can be assayed by standard colorimetric techniques. However, such a method has multiple limitations. First, the plasma of mammals contains other esterases that will hydrolyze the substrate molecule and liberate the dye (example given, acetyl cholinesterase), this enzyme assay liberates very non-specific results. Other problems with the enzymatic assay include the fact that CA is inhibited by circulating proteins, as well as drugs, including sulfanilimides, ethanol and morphine.